A small family of G protein-coupled receptor (GPCR) kinases (GRKs) negatively regulates heterotrimeric G protein signaling by phosphorylating multiple sites in the cytoplasmic loops and tails of activated GPCRs. Through this process, cells adapt to persistent stimuli that act at GPCRs and protect themselves from damage incurred by sustained signaling. GRKs can also play maladaptive roles in human disease. GRK2 is overexpressed during heart failure, which not only uncouples cardiac receptors from the central nervous system, but also promotes the release of excessive amounts of catecholamines from the adrenal gland. Inhibition of GRK2 by transgenic peptides prevents cardiac failure in mouse models, suggesting that GRK2 is an excellent target for the treatment of heart disease. However, selective small molecule inhibitors of GRKs have not been reported, perhaps due to high homology among the active sites of GRKs and other AGC kinases. Over the last six years, our lab has made significant progress in understanding the structure and function of GRKs, and we are currently investigating the molecular basis for the selective inhibition of GRK2 by a high affinity RNA aptamer. Our preliminary crystallographic studies of this complex demonstrate that the aptamer binds primarily to the large lobe of the kinase domain, where it blocks the entrance to the nucleotide binding site of the kinase domain. We hypothesize that this RNA aptamer can be used in a displacement assay to identify small molecules that bind to regions on GRK2 outside of its active site that are also critical for activity. We have designed improved versions of the original RNA aptamer for use in a robust flow cytometry protein interaction assay to screen for compounds that compete with RNA binding to GRK2. In collaboration with the Center for Chemical Genomics at the University of Michigan, we have conducted a preliminary HTS of ~40,000 compounds with excellent statistics. Using activity-based secondary screens, we will confirm which hits derived from this screen and those from screens conducted at a Molecular Libraries Probe Production Center bind directly to GRK2 and inhibit kinase activity. These compounds will be further characterized to establish membrane permeability, their mode of inhibition, and their selectivity for GRK2. Although all active molecules are of interest, small molecules that do not exhibit competitive inhibition with ATP are of particular importance because they would likely represent novel and selective therapeutic leads for the treatment of heart disease. PUBLIC HEALTH RELEVANCE: GRK2 is strongly linked to cardiovascular physiology and disease. Our flow cytometry protein interaction assay will allow us to rapidly screen large libraries of small molecules with the goal of identifying compounds that interfere with a high-affinity RNA aptamer that selectively binds to the kinase domain of GRK2. These compounds have the potential to interact with novel sites on the surface of the kinase domain and thus serve as selective inhibitors of GRK2.